We have developed a fluorogenic NAD(P)(+) detection method using 2-acetylbenzofuran. The reaction of NAD(P)(+) with 2-acetylbenzofuran produced a fluorescent product, allowing the highly-sensitive and quick detection of NAD(P)(+). This method was successfully applied to the detection of P450 substrates in the microtiter-plate format.
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http://dx.doi.org/10.1039/c3cc47264g | DOI Listing |
J Am Chem Soc
January 2025
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
We developed a single-molecule enzyme activity assay platform for NAD(P)-dependent oxidoreductases, leveraging a new NAD(P)H-responsive fluorogenic probe optimized for microdevice-based fluorometric detection. This platform enabled the detection of enzyme activities in blood and cerebrospinal fluid (CSF), including lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinases. We demonstrate its potential for activity-based diagnosis by detecting altered populations of enzyme activity species in blood and CSF from liver damage in brain tumor patients.
View Article and Find Full Text PDFJ Am Chem Soc
July 2024
Key Laboratory of Smart Drug Delivery Ministry of Education, School of Pharmacy, Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, Fudan University, Zhangheng Road 826, Shanghai 201203, China.
Ratiometric biosensors employing Förster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor-acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor () relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state.
View Article and Find Full Text PDFTalanta
December 2023
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, PR China. Electronic address:
We suggest that aggregation-induced emission (AIE) molecular probes with single charged/reactive group can exist in the formation of nanostructures but not monomers at extremely low organic solvent content. The nanoaggregates show good dispersivity and emit week emission. Stimuli-responsive assembly of nanoaggregates by electrostatic interactions can turn on the fluorescence, facilitating the design of biosensors with single-charged molecular probes as the AIE fluorogens.
View Article and Find Full Text PDFZhonghua Yi Xue Za Zhi
December 2015
Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Objective: To investigate the expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in hepatocellular carcinoma (HCC) tissues and its correlation with clinical parameters.
Method: Fluorogenic quantitative-polymerase chain reaction (FQ-PCR) was used to measure the MTHFD2 mRNA expression. The MTHFD2 protein expression was detected by immunohistochemical staining in cancerous tissues and adjacent noncancerous counterparts.
Anal Biochem
July 2014
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426 Jülich, Germany; Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, 52425 Jülich, Germany. Electronic address:
Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid.
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